Method for diagnosing immunologic food sensitivity

ABSTRACT

The invention includes novel methodology for diagnosing immunologic food or drug sensitivities. The method for diagnosing food sensitivities includes using diagnoses of other related disorders, such as microscopic colitis or other chronic immunologic/autoimmune syndromes, chronic diarrhea, irritable bowel syndrome, and hepatitis C and other hepatic diseases, Crohn&#39;s disease, alcoholism, and other idiopathic neuropsychiatric and neurologic disorders, as indicators in the diagnosis of the food sensitivity. Additionally, failure to respond to or a relapse after treatment for microscopic colitis with bismuth subsalicylate is disclosed by the present invention as being a further indicator in the diagnosis of immunologic food sensitivity. Finally, the presence of certain HLA-DQ alleles, particularly HLA-DQ1,3; -DQ1,7; -DQ1,8; and -DQ1,9, HLA-DQ1,1, and at least two subtypes of the HLA-DQ1 allele identified by molecular analysis as HLA-DQB1*0501 and HLA-DQB1*0602, as indicators in diagnosing immunologic food sensitivity, particularly gluten sensitivity or celiac sprue, and in diagnosing the related disease of microscopic colitis and other autoimmune disorders is also disclosed by the invention. A method for food sensitivity panel testing (for sensitivities other than gluten sensitivity) by detecting IgA antibodies in serum is also disclosed. A method for testing stool samples for the presence of particular antibodies, which is more sensitive and less invasive than prior art testing methods, is also disclosed for diagnosing immunologic food sensitivities. These methods of diagnosis may be used alone or in combination to further enhance the accuracy of diagnosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/670,100, which is a continuation of U.S. patent application Ser. No.09,798,557 (now U.S. Pat. No. 6,667,160). Each application is herebyincorporated by reference for all purposes.

TECHNICAL FIELD OF THE INVENTION

The invention relates to diagnosing immunologic food or drugsensitivities and their related intestinal diseases or disorders andnoninvasive testing for such. In particular, the invention relates to amethod for diagnosing immunologic food sensitivity by testing stool forthe presence of antibodies. The invention also relates to identifyingimmunologic food sensitivities, and their related intestinal diseases ordisorders, based on the presence of IgA antibodies in blood, thepresence of certain other related disorders or diseases, the presence ofcertain HLA alleles, or based on failure to respond to bismuthsubsalicylate treatment of microscopic colitis or a relapse ofmicroscopic colitis after bismuth subsalicylate treatment.

BACKGROUND OF THE INVENTION

Persons with a variety of immunologic food or drug sensitivities, andrelated intestinal diseases or disorders, may experience a number of illeffects when a particular food or drug substance, or ingredient in thefood or drug, is ingested. The ill effects from an allergy (also calledimmediate or type I hypersensitivity) to a particular food or drugsubstance or ingredient usually causes rapid development of symptomsincluding shortness of breath, hives, swelling of the mouth and throat,and sometimes abdominal pain and diarrhea. This form of food allergytraditionally has been diagnosed by skin testing or by measuring IgEantibodies in the blood directed against the food in question. However,chronic immunologic sensitivity to a food involving T and B lymphocytesas part of the chronic cell mediated immune system (also called delayedor type IV hypersensitivity) may include overt symptoms such asabdominal pain, diarrhea, constipation, and weight loss, and may alsoinclude less noticeable effects stemming from malabsorption of fluidsand dietary nutrients, such as osteoporosis, anemia, or vitamindeficiencies. Without proper diagnostic testing, which currently islimited to invasive means such as blood or serum testing or endoscopicintestinal biopsy, a person may not realize that he or she has animmunologic food sensitivity or immunologic drug sensitivity and mayunknowingly continue to consume the triggering food or drug, which mayhave long term health implications.

For purposes of this background description, the summary of theinvention, and the claims no distinction is drawn between an immunologicfood or drug sensitivity and the related intestinal disease or disorderstemming from the immune system's reaction to the food or drugingredient and they are collectively referred to as an immunologic foodsensitivity or immunologic drug sensitivity. The description provided isprimarily related to immunologic food sensitivities, but it isrecognized that certain drugs or ingredients in drugs also triggerimmunologic reactions creating an immunologic drug sensitivity similarto such a food sensitivity.

One such immunologic food sensitivity is gluten sensitivity, or moreseverely, the intestinal disease celiac sprue. Celiac sprue results froman immunologic reaction to dietary gluten contained in wheat, barley,rye, and oats, that results in any degree of intestinal histopathology.By current definitions and classic descriptions, the gluten-inducedimmunologic process causes villous atrophy and inflammation of the smallintestine, in turn, resulting in diarrhea and weight loss frommalabsorption of fluid, electrolytes, and dietary nutrients. Despite thefact that chronic diarrhea is the most common symptom of celiac sprue inits classic, villous atrophic, form, there have been no studies of theprevalence of celiac sprue or other immunologic food sensitivities inpatients presenting to physicians with chronic diarrhea or other commongastrointestinal symptoms. Additionally, there are no adequate methodsin the prior art to diagnose or noninvasively test for immunologic foodsensitivities when the patient presents with little or no commongastrointestinal symptoms or when the symptoms are also attributable toother diseases. This frequently results in either no diagnosis or themissed diagnosis of an immunologic food sensitivity.

In recent years it has been discovered that many if not most individualswith celiac sprue do not have diarrhea or weight loss, but instead haveother signs and symptoms such as vague abdominal pain, nausea, chronicfatigue, constipation, growth retardation of children, iron deficiencyanemia, osteoporosis, seizures or other neurologic disorders, orelevated liver enzyme levels in serum. Some patients may have no signsor symptoms whatsoever.

Furthermore, patients with gluten sensitivity may not have the fullydeveloped intestinal lesion associated with celiac sprue, insteadpossessing minimally inflamed or even normal small intestinal histology.Therefore, the immunologic food sensitivity of these patients may not beproperly diagnosed using known testing methods, such as endoscopicintestinal biopsy and blood or serum testing. Additionally, thesepatients may present with other immunologic diseases such as autoimmunediseases of skin, liver, joints, kidneys, pancreas, and/or thyroid amongothers, microscopic colitis, or hepatitis C-induced liver disease, whichaccording to known methodologies has furthered the misdiagnosis ofimmunologic food sensitivities in the past. However, the exactprevalence of gluten sensitivity or celiac sprue in patients presentingwith these immune disorders is unknown, and the full spectrum of suchgluten sensitivity, particularly that with fewer classic features, isnot adequately addressed in the prior art.

Traditionally, celiac sprue has been regarded as a severe malabsorptivecondition of the small intestine that presents clinically mainly withdiarrhea and significant weight loss. Although in the U.S. this conceptof the disease is still widely held, in Europe it has become recognizedthat celiac sprue does not always present in traditional fashion, butinstead may manifest with mild gastrointestinal or constitutionalsymptomatology, or asymptomatically in association with other disordersas mentioned above. For this reason, European physicians, especiallythose in geographic areas where celiac sprue has been thought to beendemic, such as Ireland, U.K., Italy, and Scandinavia, have had ahigher diagnostic index of suspicion of celiac sprue in their patientsthan American physicians have had, and consequently diagnose more casesthan traditionally has been done in American institutions. Moreover, inseveral screening studies of normal subpopulations of these Europeancountries, the disease prevalence of celiac sprue has averaged about 400per 100,000 population (1 in 250 persons).

By comparison, from limited retrospective information from a single U.S.quaternary care referral center, celiac sprue was calculated to have aprevalence of 22 per 100,000 (1 in 4000-5000) among the Americanpopulation. However, a group of 2000 blood donors from the U.S.displayed a serologic pattern suggestive of celiac sprue with afrequency of 1 in 250, similar to the prevalence of the disease inEuropean studies. Although, these blood donors were not evaluatedclinically, so that the diagnosis of celiac sprue was not formallyconfirmed, these results suggest that celiac sprue may be present in alarge number of undiagnosed Americans. Moreover, serologic data from 228individuals from a shopping mall were screened by Applicant for celiacsprue. This screening revealed one individual with both antigliadin andanti-tissue transglutaminase antibodies, which are known indicators ofgluten sensitivity or celiac sprue, who was subsequently found by smallintestinal biopsy to have a mild lesion of celiac sprue, and 28 otherswith antigliadin antibodies, without anti-tissue transglutaminaseantibodies, which is indicative of gluten sensitivity, but not the moresevere celiac sprue. Over 40% of these latter 28 patients hadsteatorrhea and mild small intestinal inflammation. These data indicatethat the prevalence of celiac sprue in the general U.S. population issimilar to that in Europe, about 1 in 225-250 persons, with milder formsof clinically important gluten sensitivity even more prevalent.

It has been known that celiac sprue occurs almost exclusively inpatients possessing certain HLA class II alleles, namely HLA-DQ2 or-DQ8. The protein products of these genes, located on the surface ofantigen presenting cells, are involved in the pathogenesis of thedisease by binding gliadin as the initial step in the inflammatoryreaction. The prevalence of these HLA genes in the general Americanpopulation, as confirmed by analysis of more than 400 normal controls,is 42% (30% for DQ2, 12% for DQ8). From these figures, a large portionof the American population is genetically capable ofgliadin-sensitivity. Thus, it is likely that celiac disease has aprevalence of 1 in 250 people in the U.S., especially considering thatthe majority of Caucasians, the race thought to be primarily affected bythe disease, living in America are descendants of European immigrants.Most of the Hispanic population of the United States also has a geneticlineage from Western Europe; however, there is no published dataregarding the prevalence of celiac sprue in this rapidly increasingsubset of the U.S. population. Preliminary data in patients with liverdisease suggests that Hispanics, at least those encountered in Texaswhich are mostly Mexican-Americans, may have the highest prevalence ofceliac sprue of any racial subset in America. Furthermore, as will bediscussed in detail below, Applicant has identified other HLA allelesand allelic combinations predisposing to gluten sensitivity or otherchronic immunologic/autoimmune diseases. The prevalence of thesegenotypes in the U.S. general population approaches 75%.

Thus, because of the sometimes protean manifestations of celiac sprue, agenerally low index of disease suspicion among the American medicalcommunity, and the commonality of the celiac-predisposing HLA genes, itis likely that gliadin sensitivity actually is relatively common, andthat mildly symptomatic celiac sprue is under diagnosed by practicingphysicians. Thus, there is a need to establish alternate and improvedmethods of diagnosing such immunologic food sensitivities so that propertreatment may be obtained.

In addition to the need to identify the correct target populations to bescreened for gluten an other food sensitivities, the most accuratemethod by which this screening should be carried out, and how and whenpositive screening tests should be followed up, also requiredetermination. The current standard by which patients are identified ashaving gluten sensitivity and the method most commonly employed toscreen asymptomatic individuals is analysis of serum for the presence ofIgG or IgA antibodies to gliadin and IgA antibodies to tissuetransglutaminase, the latter by either immunofluorescence when it iscalled antiendomysial antibody or more recently by the more objectiveELISA (enzyme-linked immunosorbent assay) method. The current standardfor diagnosing other food sensitivities is by analysis of serum for thepresence of IgE or IgG antibodies to the suspect foods, or assessing forIgE antibodies via skin prick testing. These methods are well known inthe art.

While these antibody tests, particularly antitissue transglutaminaseantibodies, are highly sensitive and specific in populations known tohave the villous atrophic form of celiac sprue, these known methods ofdiagnosis and testing, including testing coupled with endoscopicintestinal biopsy, are inadequate to diagnose patients with milder formsof gluten sensitivity and less small intestinal damage, but who stillhave troubling symptoms and/or important pathophysiologic consequences.These known tests and diagnosis methods are particularly inadequateconsidering many patients with mild gluten sensitivity do not have allor any of these antibodies in serum, compared to patients with moredeveloped intestinal damage. This is particularly problematic as themajority of gluten sensitive individuals are likely affected in thesemilder ways.

Complete knowledge is also lacking regarding the proper approach to theconverse situation, i.e., patients with antigliadin and/or antitissuetransglutaminase antibodies in serum with minimal or no detectableintestinal histopathology, most of whom have mild or no symptoms.Furthermore, the true significance of having serum antigliadinantibodies without antitissue-transglutaminase antibodies also has beenunknown. For these reasons, there has been a need to develop moresensitive and specific methods of identifying and properly diagnosingthose individuals in screened populations who have pathophysiologicconsequences of immunologic gluten sensitivity but perhaps who do notyet have fully developed celiac sprue, and hence, would have negativescreening tests by current methods and criteria.

SUMMARY OF THE INVENTION

The invention provides a method of diagnosing immunologic foodsensitivities and related diseases or disorders based on the presence ofcertain other related disorders or immunologic diseases, based on thepresence of certain HLA alleles, or based on a failure to respond tobismuth subsalicylate treatment for microscopic colitis or a relapseafter such treatment. The invention also provides a diagnostic fecaltesting method for immunologic food or drug sensitivities, as well asusing serum IgA antibodies (many of which are of intestinal origin) forfood sensitivity panel testing. Serum testing for IgA antibodiesaccording to the invention, rather than IgE or IgG antibodies under thecurrent state of the art, applies to testing for a wide range of foodsensitivities (other than gluten sensitivity, as testing for IgAantibodies to gliadin and IgA antibodies to tissue transglutaminase areknown in the art). Serum testing methods for the detection of antibodiesare well known in the art.

Although the discussion of the invention primarily relates to glutensensitivity or celiac sprue, it is recognized that these methodologiesmay be applied to identify and test for other immunologic foodsensitivities. Once such food sensitivity is sensitivity to dietaryyeast, particularly Saccharomyces cervesiae (the yeast utilized inbaker's and brewer's yeast, as well as to make fermented foods such assauerkraut and others). Other such food sensitivities includesensitivities to milk and eggs, specifically sensitivities tolactalbumin, casein, bovine serum albumin, and ovalbumin. Additionally,it is recognized that the method of the invention may be applied toidentify immunologic drug sensitivities as certain ingredients in drugstrigger immunologic reactions just as certain food substances.

DIAGNOSING IMMUNOLOGIC FOOD SENSITIVITIES AND RELATED DISEASES BASED ONTHE PRESENCE OF OTHER DISEASE OR DISORDER

In one aspect of the invention, patients at risk for immunologic foodsensitivities are identified and the immunologic food sensitivities arediagnosed based on the presence of certain other immunologic or hepaticdiseases or disorders. It is important to identify certain target groupsof the population or patients that are at higher risk of havingimmunologic food sensitivities so that these groups may be tested andproperly diagnosed and treated. Known methods for identifying anddiagnosing immunologic food sensitivities, such as serum or bloodtesting and endoscopic intestinal biopsy, and their inadequacies havebeen previously discussed. Another known method of identifyingimmunologic food sensitivities is based on the presence of symptoms;however as the symptoms of many of gastrointestinal related diseasesoverlap, an accurate diagnosis based on symptoms may be difficult.

Frequently, patients with immunologic food sensitivities also have otherrelated diseases or disorders. In such cases, treatment of the relateddisease or disorder may not be entirely effective to alleviate symptoms.There may be residual symptoms stemming from the undiagnosed anduntreated food sensitivity, rather than the treated disease or disorder.Therefore, according to the invention, an immunologic food sensitivitymay be diagnosed, and therefore properly treated, based on the presenceof certain other diseases or disorders. Additionally, according to theinvention, a patient at risk for an immunologic food sensitivity may beidentified by the diagnosis of certain other diseases or disorders,particularly those diseases or disorders that presents symptoms, such asmalabsorption of fluids or dietary nutrients, vitamin deficiency,osteoporosis, fatigue, anemia, diarrhea, weight loss, bloating,flatulence, abdominal pain, constipation, nausea, growth retardation inchildren, seizures, or other unexplained neurologic symptoms, that aresimilar to the symptoms of immunologic food sensitivities. The methodsfor testing or diagnosing the immunologic or hepatic diseases ordisorders described herein are well known in the art and these methodsare not discussed by Applicant.

As previously discussed, one such food sensitivity that is commonlyundiagnosed or misdiagnosed is gluten sensitivity, or the relateddisease celiac sprue, which is a more severe form of gluten sensitivitywith associated small intestinal damage. There is strong evidence thatcertain target groups may have a risk for gluten sensitivity or celiacsprue many times higher than asymptomatic individuals in the generalpopulation. The reason for this relates to the fact that there are many,mostly immunologic, diseases that are similarly linked to theceliac-related HLA-DQ2 and DQ8 alleles, and to the alleles identified byApplicant to predispose to gluten sensitivity; these include HLA-DQ 1,3(including the subtypes -DQ1,7, -DQ1,8, and -DQ 1,9), HLA-DQ 1,1, and atleast two subtypes of the HLA-DQ 1 allele identified by molecularanalysis as HLA-DQB1*0501 and HLA-DQB1*0602. Two examples of diseasespreviously identified to share this genetic relationship and known topose an elevated relative risk of celiac sprue are diabetes mellitustype I and Sjogren's syndrome. Furthermore, it has been shown that thelonger persons with celiac sprue go undiagnosed and continue to eatgluten, the more prevalent were other associated autoimmune diseasessuch as diabetes, arthritis, and psoriasis, among many others.

Applicant has identified microscopic colitis to be another diseaseassociated with these HLA-DQ alleles, and this immunologic colonicdisease poses a heightened concomitant risk of gluten sensitivity.Furthermore, patients presenting to medical practitioners with any oneof the number of potential signs or symptoms of active celiac sprue alsoshould have a higher prevalence of the disease than asymptomatic people.Foremost on this symptom list is chronic diarrhea; however, there havebeen no studies of the prevalence of celiac sprue in patients withchronic diarrhea. Another target population identified by Applicant ispatients carrying a diagnosis of irritable bowel syndrome or havingsymptoms of irritable bowel syndrome since the symptoms of mild celiacsprue, such as abdominal gaseous pain, occasional diarrhea, andsometimes constipation, cannot be distinguished from those of irritablebowel syndrome. Preliminary data indicates that a high percentage ofpatients diagnosed with irritable bowel syndrome have antigliadinantibodies in serum which is indicative of gluten sensitivity and celiacsprue. Somewhat unexpectedly, another patient subset identified byApplicant to have a high prevalence of gluten sensitivity by serology ispatients diagnosed with gastroesophageal reflux or havinggastroesophageal reflux symptoms. Crohn's disease patients and thosewith alcoholism were also identified by Applicant to have a greaterfrequency of gluten sensitivity than normal and other patient controlparticipants.

In a previous study carried out by the Applicant, 78 patients withtreated celiac sprue were surveyed with respect to their symptoms at thetime of their diagnosis. Eighty-three percent stated they had diarrheaprior to being diagnosed with celiac sprue. In the same study, 121randomly selected people from a Texas city's population were surveyedregarding their stool habits. One percent said they passed liquid stoolsdaily, and another 4% stated they passed loose or watery stools at leastthree times a week for greater than a continuous six-month period. Thus,chronic diarrhea probably is not rare but may be present in about 5% ofAmericans or nearly 14 million people. If even a small fraction of thischronic diarrhea is the result of dietary gluten ingestion, then a majorimpact can be made on the gastrointestinal health of Americans ifscreening tests could easily and accurately determine who may benefitfrom dietary gluten withdrawal or withdrawal of another triggeringsubstance, and practitioners applied them on a wide scale basis.

Microscopic colitis (including lymphocytic colitis and collagenouscolitis) is a chronic diarrheal syndrome associated with a normalgross/endoscopic appearance of the colon but with inflammation seen onhistologic analysis of colonic biopsies. This form of colitis is animportant cause of chronic diarrhea, accounting for 10-12% of diagnosesamong patients presenting with this symptom to university hospitals inthe U.S. Although the clinicopathologic features of microscopic colitishave been well characterized, there has previously been no realknowledge about its pathogenesis. One feature of the colonicinflammatory reaction, intraepithelial lymphocytosis, suggests that theoffending agent, whatever it may be, is presented to themucosa-associated lymphoid tissue of the colon from the luminal side. Aparallel can be drawn from the small intestinal pathologic lesion ofceliac sprue whereby antigenic protein subfractions of ingested gluten,the prototype of which is gliadin from wheat, stimulate a similarinflammatory reaction in the small intestinal mucosa. Applicant hasdetermined that the inflammatory characteristics of microscopic colitis(mononuclear inflammatory cell infiltration of the lamina propria andintraepithelial lymphocytosis) are strikingly similar to those in thesmall bowel of celiac sprue. This indicates that a common pathogenesisexists for these syndromes. Therefore, according to the presentinvention, microscopic colitis, chronic diarrhea, irritable bowelsyndrome, and gastroesophageal reflux are indicators that may be used toidentify and diagnose gluten sensitivity and other immunologic foodsensitivities. Another indicator is chronic liver disease, particularlyhepatitis C viral mechanisms, discussed below.

In addition to the histopathologic similarities of the small intestinein celiac sprue and the colon in microscopic colitis, Applicant hasdiscovered striking similarities in the HLA-DQ subtype of patients withthese two syndromes. Like the majority of patients with celiac sprue,62% of patients in a study set with microscopic colitis had HLA-DQ2 (andan additional 30% had another HLA-DQ allelic combination, namely DQ1,3).The remaining patients had HLA-DQ1,1. The HLA genetic overlap of thesetwo syndromes is one explanation of the coexistence of celiac sprue andmicroscopic colitis noted to occur in some patients. Thus, some patientswith celiac sprue have been found to have microscopic colitis beforethey eliminated gluten from their diet and others have developed colitisfollowing long periods of gluten abstinence.

Although gliadin may not be the primary etiologic factor for mostpatients with microscopic colitis (even if they possess a celiac-likeHLA type), Applicant has identified, as discussed below, thatimmunologic gluten sensitivity may cause refractoriness of the colitisto treatment and/or persistent diarrhea that continues until gluten isremoved from the diet. Thus, determining which patients with microscopiccolitis need to be treated with a gluten-free diet and on what clinicalinformation this should be based, is of practical clinical importance.The use of a diagnosis of microscopic colitis, chronic diarrhea,irritable bowel syndrome, gastroesophageal reflux, alcoholism, Crohn'sdisease, autism, neuropsychiatric syndromes, or other chronicimmunologic/autoimmune diseases as indicators to diagnose glutensensitivity or other immunologic food sensitivities, may be coupled withthe stool testing methods described below as another aspect of theinvention, to further enhance the accuracy of immunologic foodsensitivity diagnosis. Additionally, the diagnosis of an idiopathicneurologic syndrome, such as seizures, cerebellar degeneration, multiplesclerosis, peripheral neuropathy, and others, is another indicator ofgluten sensitivity or other immunologic food sensitivities that may becoupled with the stool testing methods described below to furtherenhance the accuracy of immunologic food sensitivity diagnosis.

Another clinical setting in which patients have been identified to havean elevated relative risk of celiac sprue is that of elevated hepatictransaminase levels in blood. Although it has been observed for manyyears that as many as 40% of patients with celiac sprue have elevatedliver enzymes at the time of diagnosis, it has been noted only recentlythat almost 10% of patients presenting to physicians with elevated serumliver transaminase levels of unknown etiology have celiac sprue, andmany more may have gluten sensitivity of milder degrees. Indeed, celiacsprue, which has been epidemiologically linked to certain autoimmuneliver diseases, has recently been identified to be associated withproduction of the specific autoantibody antitissue transglutaminase.

While it is possible that immunologic gluten sensitivity andinflammatory damage of the small intestine as a primary event maysecondarily cause liver inflammation, evidenced by normalization ofelevated serum transaminase levels and resolution of nonspecific hepatichistopathologic inflammation after removal of gluten from the diet inceliac sprue patients so-affected, it is also possible that certainhepatic inflammatory diseases trigger immunologic gluten intolerance andautoimmune attack on the intestine in genetically predisposedindividuals. One common hepatic disease, viral hepatitis C, inparticular has been identified to lead to secondary autoimmune processesin other parts of the body; infection with the hepatitis C virus hasbeen associated with development of antinuclear antibodies, mixedcryoglobulinemia, and lichen planus of the skin. Additionally, thecell-mediated inflammatory response of a patient with hepatitis C hasbeen linked to T cells restricted to HLA-DQ2, an allele commonlyassociated with gluten sensitivity. Through testing, Applicant has shownthat 1.2% of patients with hepatitis C have celiac sprue compared to 0%in liver control patients. Therefore, hepatitis C is a trigger for theautoimmune reactions that are pathogenic to celiac sprue.

Therefore, according to the present invention, hepatitis C and otherhepatic diseases are indicators which may be used to identify anddiagnose gluten sensitivity and other immunologic food sensitivities.Further, the use of a diagnosis of hepatitis C or other hepatic disease,as an indicator for gluten sensitivity or other immunologic foodsensitivities, may be coupled with the stool testing methods describedbelow as another aspect of the invention, to further enhance theaccuracy of immunologic food sensitivity diagnosis.

Diagnosing Immunologic Food Sensitivities and Related Diseases Based onRelapse After Treatment of Microscopic Colitis with BismuthSubsalicylate

Additionally, the use of bismuth subsalicylate (commonly available underthe trademark Pepto-Bismol, from Procter and Gamble) has beensuccessfully shown by Applicant to treat microscopic colitis.Microscopic colitis is diagnosed using methods well known in the art orusing the method of the invention described herein. According to thetreatment regimen developed by Applicant, the patient's microscopiccolitis is treated with 3 chewable tablets or swallowed caplets ofbismuth subsalicylate taken 3 times a day for about 8 weeks. Threetablespoons three times a day of the liquid form of bismuthsubsalicylate would be an equivalent dose. This amount and frequency forbismuth subsalicylate treatment is generally preferred, however otheramounts may be used depending on the particular patient being treated.Although an 8-week treatment period is preferred it is recognized thatshorter or longer treatment periods may be used.

Trials using bismuth subsalicylate have included patients with longstanding celiac sprue complicated by microscopic colitis, and patientswho were diagnosed with sprue at the time their colitis was found.Patients known to have celiac sprue, although responding to bismuthsubsalicylate treatment for their colitis, displayed a greater tendencyto relapse weeks to months after the 8-week treatment period. Although afew of the microscopic colitis patients not known to have celiac spruehave exhibited this same response-relapse pattern to bismuthsubsalicylate treatment, this response or failure to respond to the8-week treatment all together, coupled with the HLA genetic overlapbetween microscopic colitis and celiac sprue and the presence ofabnormal small bowel histology is indicative of immunologic glutensensitivity. Each of these patients' serum was analyzed for the presenceof antigliadin and antitissue transglutaminase antibodies. Only three ofthese patients had positive serum antibody tests. Therefore, relapsingor refractory colitis patients' stool was tested for the presence ofantigliadin and antitissue transglutaminase antibodies. The method fortesting the stool according to the present invention is discussed indetail below. TABLE 1 Diagnostic Parameters in 52 Patients withMicroscopic Colitis Stratified by Responsiveness to Treatment withBismuth Subsalicylate HLA-DQ2 Abnormal Serum Fecal Serum Fecal ResponsePattern n or −1,3 Small Bowel AGA IgA AGA IgA ATTA ATTA Response, norelapse 20 15 (75%)  4 (20%) 2 (15%) 15 (75%) 0 12 (60%) Response,relapse 25 25 (100%) 23 (92%) 5 (12%) 21 (84%) 1 (4%) 18 (72%) Noresponse  7  7 (100%)  7 (100%) 1 (14%)  7 (100%) 0  7 (100%)AGA—antigliadin antibodyATTA—antitissue transglutaminase antibody

Table 1 compares diagnostic parameters in the relapse patients comparedto those who responded to bismuth subsalicylate therapy without relapse.There was a greater percentage of patients with abnormal small bowelhistology and fecal antigliadin and antitissue transglutaminase IgAantibody in the relapsing/refractory groups. Based on all of these data,the 32 patients who have displayed some element of refractoriness oftheir colitis to treatment have been advised to try a gluten-free diet,including the 28 with antigliadin/antitissue transglutaminase antibodyin stool. They agreed to try the diet for at least 6 months; 24experienced a subjective improvement in their diarrhea, abdominalbloating, energy level, and general well-being and have stayed on thediet.

Thus, many patients with microscopic colitis may have mild celiac spruerequiring treatment with a gluten-free diet for complete symptom relief.Therefore, another aspect of the invention uses failure to respond to ora relapse after treatment for microscopic colitis with bismuthsubsalicylate as an indicator to diagnose some degree of celiac sprue orimmunologic food sensitivity. The use of a failure to respond or arelapse in the microscopic colitis, as an indicator for diagnosis ofgluten sensitivity or other immunologic food sensitivities, may becoupled with the stool testing methods described below, to furtherenhance the accuracy of or confirm diagnosis of an immunologic foodsensitivity.

Diagnosing Immunologic Food Sensitivities and Related IntestinalDiseases Based on HLA Gene Testing Results

As previously discussed, it is known that celiac sprue, and otherimmunologic food sensitivities and related intestinal diseases ordisorders, occur almost exclusively in patients possessing certain HLAclass II alleles, namely HLA-DQ2 or -DQ8. Methods of testing for HLAalleles are well known in the art and are not described herein. Theprotein products of these genes, located on the surface of antigenpresenting cells, are involved in the pathogenesis of the disease bybinding gliadin as the initial step in the inflammatory reaction. Theprevalence of these HLA genes in the general American population, asconfirmed by analysis of more than 400 normal controls, is 42% (30% forDQ2, 12% for DQ8).

In addition to the HLA-DQ2 or -DQ8 alleles, Applicant has discoveredthat other HLA alleles are prevalent in diseases such as microscopiccolitis, and are related to celiac sprue. In particular, HLA-DQ 1,3(including the subtypes HLA-DQ1,7, -DQ1,8, and -DQ1,9), HLA-DQ1,1, andat least two subtypes of the HLA-DQ1 allele identified by molecularanalysis as HLA-DQB1*0501 and HLA-DQB1*0602HLA-DQ1,3, are indicators ofimmunologic food sensitivity, particularly gluten sensitivity. Thedetection of these alleles, through methods well known in the prior art,is used according to the present invention as a further method ofdiagnosing immunologic food sensitivities and their related diseases,particularly gluten sensitivity, celiac sprue, and microscopic colitis.

Additionally, in Applicant's study of bismuth subsalicylate treatmentand relapse of microscopic colitis, Applicant discovered that of theseven patients with refractory microscopic colitis, all of whom hadfecal antigliadin or antitissue transglutaminase antibody detected,three patients had an HLA-DQ 1,7 type (one subtype of HLA-DQ 1,3) ratherthan DQ2. This indicates that the second most common HLA-DQ type inmicroscopic colitis, DQ1,7, is also an indicator of immunologicreactivity to gluten and other food sensitivities. Based on other data,the same is believed to be true for DQ1,8 and DQ1,9, all of which aresubtypes of DQ1,3.

Therefore, it is another aspect of the invention to use prior artmethods to detect the presence of HLA-DQ 1,3 (including the subtypesHLA-DQ1,7, -DQ1,8, and -DQ1,9), HLA-DQ1,1, and at least two subtypes ofthe HLA-DQ 1 allele identified by molecular analysis as HLA-DQB1*0501and HLA-DQB1*0602 wherein the detection of any of these alleles isutilized as an indicator in the diagnosis of immunologic foodsensitivity and related disease, particularly gluten sensitivity, celiacsprue, or microscopic colitis. The use of these alleles, as diagnosticindicators for gluten sensitivity or other immunologic foodsensitivities, may be coupled with the stool testing methods describedbelow, to further enhance the accuracy of immunologic food sensitivitydiagnosis.

Diagnosing Immunologic Food or Drug Sensitivity by Testing Fecal Samplesfor Antibodies

The following discussion relates to testing for gluten sensitivity orceliac sprue, as gluten sensitivity and celiac sprue are perhaps themost commonly under diagnosed or misdiagnosed food sensitivities. It isrecognized that the method described is equally applicable to other foodor drug sensitivities, with the exception that the antibody beingscreened for varies depending on the particular food or drug sensitivityunder diagnosis.

Clinicians and clinical researchers who frequently are asked to evaluatepatients with suspected gluten intolerance are quite familiar withindividuals who give compelling histories regarding gastrointestinalsymptoms provoked by gluten-containing foods, which abate uponwithdrawal from gluten, but who have no detectable evidence of celiacsprue by serology or intestinal histology. This scenario has been calledgluten-sensitive diarrhea, or more simply, gluten sensitivity. Althoughsuch individuals indeed have a mild form of gluten intolerance, whichmay be due simply to the poor digestability of wheat, there are similarindividuals who actually have an element of immunologic reactivity togluten, i.e., serum and now fecal antigliadin antibodies, but who do notmanifest all the features diagnostic of celiac sprue, namelyantiendomysial or antitissue transglutaminase antibodies, or a fullydeveloped small intestinal lesion.

Traditionally, screened individuals or patients with isolatedantigliadin antibodies in serum were either not biopsied to prove ordisprove that they had celiac sprue, or had an intestinal biopsy thatwas normal or only minimally inflamed and hence were not diagnosed withor treated for celiac sprue. These known approaches result in nodiagnosis or a misdiagnosis of immunologic food sensitivities.Therefore, the present invention provides a more sensitive method oftesting intestinal physiologic function to be certain that theantigliadin humoral response is not associated with occult intestinaldamage, symptoms, and/or pathophysiological consequences, some of whichmay be irreversible, such as short stature and osteopenia.

A stool sample with isolated antigliadin antibodies indicates that thepatient suffers from some degree of immunologic gluten sensitivity ormild celiac sprue, regardless of the results of the serum test. Suchpatients should avoid ingestion of dietary gluten or other triggeringfood substance depending on the particular immunologic food sensitivityantibody being tested.

Approximately 10% of randomly selected individuals from the “normal”population have antigliadin antibodies detected in serum. For no logicalreason except that such individuals were considered “normal,” this hasbeen interpreted to mean that antigliadin antibodies per se have no realclinical significance. However, an alternative interpretation of thisscenario is that the 90% without antigliadin antibodies in serum aretruly normal with respect to immunologic gliadin reactivity, and thatthe 10% that possess serum antigliadin antibodies are abnormal relativeto the majority. Furthermore, there have been no extensive physiologicand histopathologic studies of patients with isolated antigliadinantibody in serum.

An evaluation of stool by Applicant in such individuals revealssteatorrhea in 43%, and an abnormal small intestinal permeability basedon a sucrose absorption test in 56%, even though upper small intestinalhistology is normal or only minimally inflamed. This indicates thatantigliadin antibody production is abnormal and the gluten sensitivityin such individuals may be associated with intestinal symptoms and/ordamage. The same can be said about isolated antigliadin antibodies foundin stool, according to the testing methodology discussed below. Thusanother aspect of the invention is that isolated presence of antigliadinantibodies (IgG or IgA) detected in serum or stool indicates thatclinically important gluten sensitivity is present.

Although about two-thirds of patients with microscopic colitis have aceliac-predisposing HLA class II allele and some degree of smallintestinal histopathology, only 13% and 5%, respectively, havedetectable antigliadin IgA and antitissue transglutaminase IgA antibodyin serum and with low titers. As these IgA antibodies are produced inthe intestinal mucosa and secreted into the lumen, a greater percentageof patients may have these antibodies detectable in intestinal effluent,i.e., stool. Using a modified version of an FDA approved ELISA kit,which is well known in the art and commercially available from INOVADiagnostics, San Diego, Calif., patients with celiac sprue, refractorysprue and microscopic colitis, the microscopic colitis syndrome, andnormal volunteers were tested for the presence of antigliadin andantitissue transglutaminase IgA antibody in the stool. The modificationof the kit's published method first utilizes stool rather than serum,and utilizes methods of concentrating the stool for enhanced detectionof these antibodies. The stool sample is collected after excretion bythe patient in an appropriate specimen container.

Applicant's sample collection kit, including instructions and a specimencontainer, may be order by the patient through Applicant's laboratory,Enterolab. Preferably, the kit is ordered on-line via Applicant's website on the Internet. Once shipped to the patient, the patient collectsa stool sample in the specimen container according to the instructionsprovided, which may include instruction regarding changes to thepatient's diet for a period of time prior to sample collection. Thesample is then returned to the lab for testing via the air shippinglabel and packaging provided. Although this is the preferred method forcollecting a sample from a patient, it is recognized that other methodsof obtaining a sample may be used within the scope of the invention.Such methods include having the patient physically go to a clinic, lab,doctor's office, or other appropriate medical establishment to provide astool sample. Additionally, if the clinic, lab, or doctor's office islocal relative to the patients' residence, the patient may pick-upinstructions and a specimen container for collection of the sample athome and then return the sample for testing.

Once collected and received by the lab, the fecal matter of the sampleis then concentrated and screened using the ELISA kit to detect theappropriate antibody based on the particular food sensitivity underdiagnosis. With the exception of modifying the handling of the sampleprior to testing as described below, the standard method of the ELISAkit is utilized to screen the sample to detect antibodies. The detectionof a particular antibody then forms the basis for a diagnosis of thefood sensitivity associated with that antibody, for instance thedetection of antigliadin IgA or antitissue transglutaminase IgA resultsin a diagnosis of gluten sensitivity or celiac sprue.

One method of concentrating the stool specimen is to spin the stool in acentrifuge to obtain a watery supernatant. The rotation of thecentrifuge is preferably between 13,500 rpm and 20,000 rpm, but otherrotation speeds may be used depending on the viscosity of the specimen.The modification of the test kit method involves using the supernatantas an undiluted test specimen, rather than diluting the specimen withthe diluent provided in the test kit. Although it is preferred that thespecimen be analyzed undiluted, reconstituted lyophilized samples mayalso be used. The undiluted specimen is added to the test well in anamount approximately equal to the required volume of diluted serum. Thesample is then tested for the appropriate antibody, depending on theimmunologic food sensitivity being considered for diagnosis.

Another method of concentrating the stool specimen, which isparticularly used when the specimen is watery or diarrheal, is tofreeze-dry or lyophilize the specimen to solid material andreconstituting it with water at a dry matter to water ratioapproximately equal to that of non-diarrheal stool. This dry matter towater ratio is preferably approximately 25% dry matter to 75% water,although other ratios may be used. The sample is then placed in the testwell and tested for the appropriate antibody. Compared to proper normalcontrols, this concentration method enhances the detection of glutensensitivity in a suspect target diarrheal population, such asmicroscopic colitis, by approximately 50%, increasing diagnosis throughpositive testing from 50% to 75%.

The upper limit of “normal” for fecal antigliadin IgA was derived fromthe mean +2SD of values measured in stools obtained from 13 normalvolunteers; the upper limit of “normal” for fecal antitissuetransglutaminase was calculated in the same manner. The upper limit for“normal” antigliadin IgA is about 10 units and the upper limit for“normal” antitissue transglutaminase IgA is about 10 units.

Table 2 shows the results of this testing. In the 12 normal volunteers,25% of the individuals' stools were positive for antigliadin IgAantibody, which indicates that these individuals have undiagnosed glutensensitivity. “Normal” volunteers with symptoms had an even higherdetection rate. Untreated celiac sprue patients had a high percentage,approximately 76%, of antigliadin IgA antibody in serum; while 100% hadthese antibodies detected in stool. In contrast, patients withrefractory sprue or microscopic colitis, had a much higher detectionrate of these antibodies in stool compared to the almost non-existentpresence in serum. Supportive of the test's accuracy is that treatedceliac sprue patients who do not eat gluten and have no symptoms, hadthe lowest positivity rate. Fecal antigliadin IgG antibody wasundetectable in all patients (data not shown in table), supporting thefact that the fecal IgA antibodies are secreted into the intestinerather than nonspecifically leaking from the serum (because if it was aleak of serum antibody into the intestine, IgG antigliadin antibody alsowould have been detected in stool). Therefore, in one aspect of theinvention stool samples are tested for antibodies to detect and diagnoseimmunologic food sensitivities. TABLE 2 Antigliadin IgA AntibodyDetection Rates in Serum vs. Stool Serum Fecal AGA in Group Tested n AGAIgA AGA IgA stool, not serum Untreated celiac 17 13 (76%) 17 (100%) 4(24%) Refractory 4  1 (25%)  4 (100%) 3 (75%) Microscopic Colitis 46  5(11%) 35 (76%)  30 (65%)  Normal Volunteers 12 0 (0%) 3 (25%) 3 (25%)Symptomatic Non- 8 0 (0%) 6 (75%) 6 (75%) patients Treated Celiac Sprue11  2 (18%) 1 (9%)  1 (9%)  MC/Treated Sprue 6 0 (0%) 4 (67%) 4 (67%)AGA—antigliadin antibody

Further, all patients with microscopic colitis and fecal antigliadin andantitissue transglutaminase antibodies had some degree of smallintestinal histopathology. In fact, one of these patients, and the onewith the most abnormal celiac-like small intestinal biopsies observed ina patient with microscopic colitis, was a middle-aged woman with HLA-DQ2and fecal antigliadin antibody but no antigliadin orantiendomysial/antitissue transglutaminase antibodies in her serum. Thisparticular patient had gross evidence of villous atrophy, i.e.,scalloping of duodenal folds, visualized at the time of her endoscopy.

Recently fecal samples from patients with chronic diarrhea were testedfor celiac sprue. Additionally, antigliadin and antitissuetransglutaminase antibody tests, which are well known in the art, wereused to screen serum in 50 patients with chronic diarrhea and stool inan additional 46. Table 3 shows the results of this analysis. Whilethere was a relative paucity of antigliadin and antitissuetransglutaminase antibody test positivity from analysis of serum, arelatively large percentage of patients also had these antibodiesdetected in stool. Thus, like patients with microscopic colitis,analysis of stool may be more sensitive than tests of serum fordetection of antigliadin and antitissue transglutaminase IgA antibodies.

Another antibody detected in stools of patients is an antibody to theyeast present in common baked goods and beer. The yeast is calledSaccharomyces cervesiae. This, as well as sensitivities to other foodsubstances such as cow's milk (whereby antibodies to lactalbumin,casein, and bovine serum albumin are detected) and chicken's eggs(antibodies to ovalbumin), is another immunologic food sensitivitydiagnosable by the method of the invention. TABLE 3 Frequency of CeliacSprue Serologic Test Positivity in Patients with Chronic Diarrhea AGAIgG ATTA AGA IgG AGA IgA or IgA IgA Serum Screened 6 (12%) 2 (4%)  8(16%) 1 (2%) (n = 50) Stool screened 0 19 (41%) 19 (41%) 10 (22%) (n =46)AGA—antigliadin antibodyATTA—antitissue transglutaminase antibody

The stool sample is preferably tested according to the invention byusing the following steps. The stool is homogenized by hand mixing,although it is recognized that other mechanical methods of mixing may beutilized. The stool sample is concentrated, preferably using one of thepreviously described methods, however, other methods of concentrationmay be used within the scope of the invention. An aliquot ofapproximately 20 grams is preferably used as a sample, however it isrecognized that other sample sizes may be used. The supernatant will beanalyzed for antigliadin and antitissue transglutaminase IgA antibodies,or other appropriate antibodies according to the particular immunologicfood or drug sensitivity under diagnosis.

The ELISA serum testing kit, which is also well known in the art, may beutilized to test the stool supernatant. It is preferred that thesupernatant be analyzed undiluted through the modification of well-knownserum testing methods described. However, reconstituted lyophilizedsamples may also be used according to the invention. The presence ofthese antibodies, or other immunologic food sensitivity triggeredantibodies depending on which particular food sensitivity is beingtested, indicates that the patient does have the particular immunologicfood sensitivity. The patient should be treated accordingly by removingthe triggering substance from his or her diet or by other methods knownin the art.

Although specific parameters and equipment have been discussed in thisaspect of the invention, it is understood that the parameters may differand that different equipment may be used to carry out the disclosedmethodologies without deviating from the scope of the invention.

This stool testing method may be combined with one or more of theimmunologic food sensitivity diagnosis indicators previously describedaccording to the invention in order to further enhance the sensitivityand accuracy of immunologic food sensitivity diagnosis.

1. A method for diagnosing an immunologic food sensitivity comprisingthe steps of: collecting a fecal sample; screening the fecal sample todetect the presence of an antibody to a particular food substance; anddiagnosing an immunologic food sensitivity based on the presence of theantibody.
 2. The method of claim 1 further comprising the step ofconcentrating the fecal sample to obtain a testing portion after saidcollecting step and wherein said testing portion is the sample in saidscreening step.
 3. The method of claim 2 wherein the testing portion isundiluted.
 4. The method of claim 2 further comprising the step ofhomogenizing the fecal sample prior to said concentrating step.
 5. Themethod of claim 2 wherein the testing portion is about 20 grams.
 6. Themethod of claim 1 wherein said screening step utilizes an enzyme-linkedimmunosorbant assay (ELISA) testing kit to detect the presence of anantibody to a particular food substance.
 7. The method of claim 2wherein said screening step utilizes an enzyme-linked immunosorbantassay (ELISA) testing kit to detect the presence of an antibody to aparticular food substance.
 8. The method of claim 2 wherein saidconcentrating step comprises the steps of: centrifuging the fecalsample; removing a supernatant portion from the centrifuged fecalsample; and using the supernatant portion as the testing portion.
 9. Themethod of claim 8 wherein the centrifuging step is performed at arotation speed dependant upon the viscosity of the sample.
 10. Themethod of claim 8 wherein the rotation speed is between about 13,500 and20,000 rpm.
 11. The method of claim 8 wherein the testing portion isscreened undiluted and is about equal to an amount of diluted serumrequired by standard use of an ELISA kit.
 12. The method of claim 2wherein said concentrating step comprises the steps of: freeze-dryingthe fecal sample to a solid material; and reconstituting the solidmaterial with water to form a reconstituted testing portion.
 13. Themethod of claim 12 wherein said reconstituted testing portion is about25% solid material and about 75% water.
 14. The method of claim 2wherein said fecal sample contains more than about 90% water in itsexcreted state and wherein said concentrating step comprises the stepsof: freeze-drying the fecal sample to a solid material; andreconstituting the solid material with water to form a reconstitutedtesting portion.
 15. The method of claim 1 wherein the immunologic foodsensitivity is gluten sensitivity or celiac sprue.
 16. The method ofclaim 15 wherein the antibody is antigliadin IgA or antitissuetransglutaminase IgA.
 17. The method of claim 2 wherein the immunologicfood sensitivity is gluten sensitivity or celiac sprue.
 18. The methodof claim 17 wherein the antibody is antigliadin IgA or antitissuetransglutaminase IgA
 19. The method of claim 1 wherein the immunologicfood sensitivity is yeast sensitivity.
 20. The method of claim 2 whereinthe immunologic food sensitivity is yeast sensitivity.